JOURNAL OF FERMENTATION AND BIOENGINEERING Vol.70,N0.5,317-321.1990 Enhanced Production of Shikonin by Lithospermum erythrorhizon Cells Immobilized in Polyurethane Foam Matrices YOUNG HOON PARK,1*WEON TAEK SEO,'AND JANG RYOL LIU2 Biochemical Process Laboratory and Plant Cell Biology Laboratory,?Genetic Engineering Center,The Korea Institute of Science and Technology,P.O.Box 17,Daeduk Science Town,Taejon 305-333,Korea Received 18 April 1990/Accepted 11 September 1990 Production of shikonin by Lithospermum erythrorhizon cells immobilized in polyurethane foam matrices was investigated.The cells were effectively entrapped inside the polymer matrices prepared in a cubic form with a lateral length of 0.5 cm.They proliferated further by incubation and resulted in immobilized polymer foam cubes which were fully packed with cell aggregates.Shikonin production was observed to increase significantly when the immobilized cells were used.It was excreted by the cells and directly adsorbed,by hydrophobic inter- action,to the polymer site.The product could be recovered,after the cells were separated,by direct solvent extraction from the shikonin-adsorbed polyurethane matrices.It is more interesting to note that a single-stage culture with immobilized L.erythrorhizon cells yielded higher shikonin productivity than the conventional two-stage culture system,which is considered to be of practical importance for industrial applications. Plant cell culture technology has been developing rap- tion.With the cell line used in this study,SH medium was idly since the 1970s,and successfully applied to the produc- found to be superior for cell growth to other media like tion of many high value-added chemicals.Among them, MG-5 described by Fujita et al.(6).Modifications of the shikonin has been reported as the most successful example SH medium were made;SH medium containing 2 mg//of for the mass production of plant secondary metabolites. p-chlorophenoxyacetic acid and 0.1 mg//of kinetin was Large scale production of shikonin has been carried out by designated as SHCK medium,and that with 1.75 mg//of a two-staged culture method using the improved media indole-3-acetic acid and 0.1 mg//of kinetin as SHIK me- MG-5 and M-9 for cell growth and for product forma- dium.The cell line was maintained by regular subcultures tion,respectively (1). in SHCK liquid and agar media every eight days. Immobilization techniques have also been successfully Support material A sheet of polyurethane foam (Sam applied to plant cell culture systems;entrapment of grow- Kwang Chemical Co.,Seoul,Korea)was cut into cubic ing plant cells within inert polysaccharides(2)and synthe- forms with a lateral length of 0.5 cm.The foam matrices tic resin polymers (3),and their adsorption to porous poly- had a porosity of around 0.97 and pore size of 40 pores per mer materials(4)are typical examples.By immobilization. linear inch.Polyurethane foam cubes prepared in such a plant cells are protected from external environmental fac- manner were washed several times with ethanol and distilled tors such as shear stress,and the cellular activity itself can water and then dried before they were used. be further enhanced by facilitated cell-to-cell contact.The Cell cultivation in shake flasks Cells of L.ery- possibility of ease of scale-up and of improvement in proc. throrhizon were cultivated in 250 ml Erlenmeyer flasks ess productivity resulting from employing immobilized containing 40 ml liquid medium in a gyratory shaking in- cells should therefore be carefully considered in any plant cubator at 25C and 100 rpm under dark condition.After cell culture system in order to achieve proper economic 9d of cultivation,cells were inoculated to a specified cul- feasibility. ture medium.10%(v/v)inoculum was used for a single- In the present work,polyurethane foam was selected as stage culture,or for the first stage of a two-stage culture a support material for immobilization of Lithospermum system.In the two-stage culture,the cells precultured in erythrorhizon cells to produce shikonin derivatives.Cell the first stage were harvested on a stainless steel mesh growth and shikonin production were examined by using (mesh size,ca.30 um)and transferred to the second-stage the immobilized plant cells.Considerable improvements flask containing a specified medium. were observed in the process productivity of the plant cell Immobilization of the cells Polyurethane foam culture system,and the results are reported in this paper. cubes prepared as above were added to the culture flask such that their total weight constituted 1%(w/v)of the MATERIALS AND METHODS culture broth and then sterilized at 120C for 15 min. Since each foam cube weighed approximately 0.0025- Cell line and culture media All experiments were per- 0.0030 g,the content was roughly estimated to be about formed using L.erythrorhizon KCTC PCL 52001.which 4,000 cubes per liter of culture broth.10%(v/v)inoculum was obtained from Korean Collections for Type Cultures was then made with free cells of L.erythrorhizon Genetic Engineering Center,The Korea Institute of cultivated as above such that the flask contained a cell con- Science and Technology,Taejon,Korea. centration of ca.40 g of fresh cell weight//.When the flask The basal media used were Shenk and Hildebrandt (SH) was incubated on a gyratory shaker,the cells became en- medium(5)for cell growth and M-9(6)for product forma- trapped in the pores of the polyurethane foam.It was ob- served that the cells were almost completely entrapped in Corresponding author. the pores within 2-3 d of incubation.Since free cells of L. 317JOURNAL OF FERMENTATION AND BIOENGINEERING VOI. 70, No. 5, 317-321. 1990 Enhanced Production of Shikonin by Lithospermum erythrorhizon Cells Immobilized in Polyurethane Foam Matrices YOUNG HOON PARK, 1. WEON TAEK SEO, 1 AND JANG RYOL LIU 2 Biochemical Process Laboratory ~ and Plant Cell Biology Laboratory, 2 Genetic Engineering Center, The Korea Institute of Science and Technology, P.O. Box 17, Daeduk Science Town, Taejon 305-333, Korea Received 18 April 1990/Accepted 11 September 1990 Production of shikonin by Lithospermum erythrorhizon cells immobilized in polyurethane foam matrices was investigated. The cells were effectively entrapped inside the polymer matrices prepared in a cubic form with a lateral length of 0.5 cm. They proliferated further by incubation and resulted in immobilized polymer foam cubes which were fully packed with cell aggregates. Shikonin production was observed to increase significantly when the immobilized cells were used. It was excreted by the cells and directly adsorbed, by hydrophobic inter￾action, to the polymer site. The product could be recovered, after the cells were separated, by direct solvent extraction from the shikonin-adsorbed polyurethane matrices. It is more interesting to note that a single-stage culture with immobilized L. erythrorhizon cells yielded higher shikonin productivity than the conventional two-stage culture system, which is considered to be of practical importance for industrial applications. Plant cell culture technology has been developing rap￾idly since the 1970s, and successfully applied to the produc￾tion of many high value-added chemicals. Among them, shikonin has been reported as the most successful example for the mass production of plant secondary metabolites. Large scale production of shikonin has been carried out by a two-staged culture method using the improved media MG-5 and M-9 for cell growth and for product forma￾tion, respectively (1). Immobilization techniques have also been successfully applied to plant cell culture systems; entrapment of grow￾ing plant cells within inert polysaccharides (2) and synthe￾tic resin polymers (3), and their adsorption to porous poly￾mer materials (4) are typical examples. By immobilization, plant cells are protected from external environmental fac￾tors such as shear stress, and the cellular activity itself can be further enhanced by facilitated cell-to-cell contact. The possibility of ease of scale-up and of improvement in proc￾ess productivity resulting from employing immobilized cells should therefore be carefully considered in any plant cell culture system in order to achieve proper economic feasibility. In the present work, polyurethane foam was selected as a support material for immobilization of Lithospermum erythrorhizon cells to produce shikonin derivatives. Cell growth and shikonin production were examined by using the immobilized plant cells. Considerable improvements were observed in the process productivity of the plant cell culture system, and the results are reported in this paper. MATERIALS AND METHODS Cell line and culture media All experiments were per￾formed using L. erythrorhizon KCTC PCL 52001, which was obtained from Korean Collections for Type Cultures, Genetic Engineering Center, The Korea Institute of Science and Technology, Taejon, Korea. The basal media used were Shenk and Hildebrandt (SH) medium (5) for cell growth and M-9 (6) for product forma- * Corresponding author. 317 tion. With the cell line used in this study, SH medium was found to be superior for cell growth to other media like MG-5 described by Fujita et al. (6). Modifications of the SH medium were made; SH medium containing 2 mg/l of p-chlorophenoxyacetic acid and 0.1 mg/l of kinetin was designated as SHCK medium, and that with 1.75 mg/l of indole-3-acetic acid and 0.1 mg/l of kinetin as SHIK me￾dium. The cell line was maintained by regular subcultures in SHCK liquid and agar media every eight days. Support material A sheet of polyurethane foam (Sam Kwang Chemical Co., Seoul, Korea) was cut into cubic forms with a lateral length of 0.5 cm. The foam matrices had a porosity of around 0.97 and pore size of 40 pores per linear inch. Polyurethane foam cubes prepared in such a manner were washed several times with ethanol and distilled water and then dried before they were used. Cell cultivation in shake flasks Cells of L. ery￾throrhizon were cultivated in 250 ml Erlenmeyer flasks containing 40 ml liquid medium in a gyratory shaking in￾cubator at 25°C and 100 rpm under dark condition. After 9 d of cultivation, cells were inoculated to a specified cul￾ture medium. 10~ ~ (v/v) inoculum was used for a single￾stage culture, or for the first stage of a two-stage culture system. In the two-stage culture, the cells precultured in the first stage were harvested on a stainless steel mesh (mesh size, ca. 30/zm) and transferred to the second-stage flask containing a specified medium. Immobilization of the cells Polyurethane foam cubes prepared as above were added to the culture flask such that their total weight constituted 1% (w/v) of the culture broth and then sterilized at 120°C for 15min. Since each foam cube weighed approximately 0.0025- 0.0030 g, the content was roughly estimated to be about 4,000 cubes per liter of culture broth. 10% (v/v) inoculum was then made with free cells of L. erythrorhizon cultivated as above such that the flask contained a cell con￾centration of ca. 40 g of fresh cell weight/l. When the flask was incubated on a gyratory shaker, the cells became en￾trapped in the pores of the polyurethane foam. It was ob￾served that the cells were almost completely entrapped in the pores within 2-3 d of incubation. Since free ceils of L